1. Field of the Invention
The present invention relates to a lyophilized reagent for polymerase chain reaction, more specifically, to a lyophilized reagent for DNA polymerase chain reaction which is stabilized and lyophilized to ease DNA amplification, sequencing and diagnosis of disease.
2. Description of the Prior Art
DNA polymerase chain reaction (hereinafter referred to as "PCR") allows the DNA sequence at a specific region of a genome to be amplified by more than a million-fold, provided that at least part of its nucleotide sequence is already known. Portions of the sequence that surround the region to be amplified are used to design two synthetic DNA oligonucleotides, one complementary to each strand of the DNA double helix. These oligonucleotides serve as primers for in vitro DNA synthesis, which is catalyzed by a DNA polymerase, and they determine the ends of the final DNA fragment that is obtained. Each cycle of the PCR requires denaturation to separate two strands of the DNA double helix, annealing for specific hybridization to complementary DNA sequences, and extension for synthesis of DNA. For effective amplification, 30 to 40 cycles of reaction are required.
In conclusion, the PCR by which a specific nucleotide sequence can be amplified in vitro from the genomic DNA, enables the detection of a specific DNA sequence and the acquisition of the DNA fragment of interest in a large quantity in a short period of time.
The PCR technique has been utilized in a wide range of life sciences, such as in the detection of genes associated with genetic diseases (see: Suzuki, Y. et al., Anal. Biochem., 192:82-84 (1991); Gibbs, R. A. et al., Nucleic Acids Res., 17:2374-2448 (1989); Ballabio, A. et al., Nature, 343:220 (1990)); in the detection and expression of a specific MRNA by way of cDNA amplification by employing reverse transcription-PCR (RT-PCR) and RACE (rapid amplification of cDNA end) methods (see: Rappolee, D. A. et al., Science, 241:708-712 (1991); Frohman, M. A. et al., Proc. Natl. Acad. Sci., U.S.A., 85:8998-9002 (1988)); in direct nucleotide sequencing from the amplified product of DNA (see: Gyllensten, U. B. et al., Proc. Natl. Acad. Sci., U.S.A., 85:7652-7657 (1988)); in the analysis of VNTR (various number of tandem repeat) (see: Ali, S. et al., Nucleic Acids Res., 16:8487-8496 (1988)); and, in genetic mapping (see: Nelson, D. L. et al., Proc. Natl. Acad. Sci., U.S.A., 86:6686-6690 (1989)). In addition, it has been also utilized in the diagnosis of a variety of diseases, such as HTLV-I (human T-cell lymphoma/leukemia virus type I) (see: Kwok, S. et al., Blood, 72:1117-1123 (1988)), HIV (human immunodeficiency virus) (see: Ou, C. Y. et al., J. Infect. Dis., 158:1170-1176 (1988)) and HBV (hepatitis B virus) (see: Kaneko, S. et al., Proc. Natl. Acad. Sci., U.S.A., 86:312-316 (1989)), and currently its usefulness becomes more and more widespread.
Furthermore, applications of said PCR technique, e.g., DD-PCR (differential display-PCR) and Immuno-PCR (see: Hong, Z. et al., Nucleic Acids Res., 21:6038-6039 (1993)) have been developed, which permit detection of only a small portion of RNA or DNA in question not detectable using currently available methods.
In the amplification of nucleic acid by PCR technique, every component of the reaction mixture for PCR, i.e., a template DNA, primers, reaction buffer, MgCl.sub.2, KCl, dNTPs (dATP, dCTP, dGTP and dTTP) and DNA polymerase, must be mixed in step-wise fashion or simultaneously at the initial step, prior to initiating the reaction. Accordingly, it has been cumbersome to add and mix the trace amounts of each component in a separate manner for every test sample, so experimental errors have been frequently accompanied. Especially when numerous samples are to be analyzed in a short period of time, the inefficiency and experimental errors accompanied have become serious obstacles in the experiments.
Moreover, it has been also known that the aerosol which develops when sample loading buffer is added to the PCR product, frequently induces carry-overcontamination (see: Kwok, S. et al., Nature, 339:237-238 (1989)) and leads to a false positive response, which has been an important problem to be solved, especially when used in diagnosis of diseases.